Pseudomycin natural products

ABSTRACT

The invention relates to pseudomycin natural products including pseudomycins A′ and B′, methods for making such pseudomycins, and methods employing antifungal activity of these pseudomycins. NMR and mass spectrometry indicate formula (I) for pseudomycin A′. NMR and mass spectrometry indicate formula (II) for pseudomycin B′.

FIELD OF THE INVENTION

[0001] The present invention relates to pseudomycin natural productsincluding pseudomycins A′ and B′, methods for making such pseudomycins,and methods employing antifungal activity of these pseudomycins.

BACKGROUND

[0002] Fungal infections are a significant cause of disease, degradationof quality of life, and mortality among humans, particularly for immunecompromised patients. The incidence in fungal infections in humans hasincreased greatly in the past 20 years. This is in part due to increasednumbers of people with immune systems weakened or devastated by organtransplants, cancer chemotherapy, AIDS, age, and other similar disordersor conditions. Such patients are prone to attack by fungal pathogensthat are prevalent throughout the population but are kept in check by afunctioning immune system. These pathogens are difficult to controlbecause some existing antifungal agents are either highly toxic or onlyinhibit fungal activity. For example, the polyenes are fungicidal buttoxic: whereas, the azoles are much less toxic but only fungistatic.More importantly, there have been recent reports of azole and polyeneresistant strains of Candida which severely limits therapy optionsagainst such strains.

[0003]Pseudomonas syringae produce several classes of antifungal orantibiotic agents, such as the pseudomycins, syringomycins,syringotoxins, and syringostatins, which are lipodepsinonapeptides.Natural strains and transposon generated mutants of P. syringae producethese lipodepsinonapeptides. Several of the pseudomycins, syringomycinsand other lipodepsipeptide antifungal agents have been isolated,chemically characterized, and shown to possess wide spectrum antifungalactivity, including activity against important fungal pathogens in bothhumans and plants. For example, pseudomycins A, B, C and C′ have eachbeen isolated and purified and their structures have been characterizedby methods including amino acid sequencing, NMR, and mass spectrometry.See, e.g. Ballio et al., “Novel bioactive lipodepsipeptides fromPseudomonas syringae: the pseudomycins.” FEBS Lett. 355, 96-100 (1994)and U.S. Pat. No. 5,576,298, The pseudomycins, the syringomycins, thesyringotoxins, and the syringostatins represent structurally distinctfamilies of antifungal compounds.

[0004] None of the pseudomycins, syringomycins, syringotoxins, orsyringostatins has been brought to market for antifungal therapy.Discovery of undesirable side effects, making formulations, scaling upproduction, and other development problems have thus far preventedexploitation of the pseudomycins, syringomycins, syringotoxins, orsyringostatins against the full range of fungal infections that affectanimals, humans and plants. There remains a need for an antifungal agentthat can be used against infections not treated by existing antifungalagents and for application against infections in animals, humans, orplants.

SUMMARY OF THE INVENTION

[0005] The present invention provides a pseudomycin natural productproduced by P. syringae. The pseudomycin natural product includes adepsinonapeptide ring with the sequenceSer-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr, more specifically,L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl), withthe carboxyl group of the ClThr and the hydroxyl group of the serineclosing the ring with a lactone bond. Pseudomycin A′ (IA) includes a3,4dhydroxypentadecanoic acid moiety, the carboxyl group of which formsan amide bond with the amine group of the N-terminal serine.

[0006] Pseudomycin B′ (IB) includes a 3-hydroxydodecanoic acid moiety,the carboxyl group of which forms an amide bond with the amine group ofthe N-terminal serine.

[0007] The invention also relates to methods employing a pseudomycinnatural product, such as pseudomycin A′, pseudomycin B′ or a mixturethereof, for inhibiting fungal activity or for reducing the symptoms ofa fungal infection in a patient in need thereof. Such methods can killthe fungus, decrease the burden of a fungal infection, reduce feverand/or increase the general well being of a patient. The methods of theinvention are effective against fungi such as Candida parapsilosis,Candida albicans, Cryptococcus neoformans, and/or Histoplasmacapsulatum.

DETAILED DESCRIPTION

[0008] Pseudomycins

[0009] As used herein, pseudomycin or pseudomycin natural product refersto one or more members of a family of antifungal agents that has beenisolated from the bacterium Pseudomonas syringae. A pseudomycin is alipodepsipeptide, a cyclic peptide including one or more unusual aminoacids and having one or more appended hydrophobic or fatty acid sidechains. Specifically, the pseudomycins are lipodepsinonapeptides, with acyclic peptide portion closed by a lactone bond and including theunusual amino acids 4-chlorothreonine, 3-hydroxyaspartic acid,dehydro-2-aminobutyric acid, and 2,4-diaminobutyric acid. It is believedthat these unusual amino acids are involved in biologicalcharacteristics of the pseudomycins, such as stability in serum andtheir killing action.

[0010] Each pseudomycin has the same cyclic peptide nucleus, but theydiffer in the hydrophobic side chain attached to this nucleus. Eachpseudomycin has a cyclic nonapeptide ring having the sequenceSer-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr (i.e., Serine:2,4-Diaminobutyric acid: Aspartic acid: Lysine, 2,4Diaminobutyric acid:alloThreonine; Dehydro-2-aminobutyric acid; 3-hydroxy Aspartic acid:4-chloroThreonine), with the carboxyl group of the ClThr and thehydroxyl group of the serine closing the ring with a lactone bond. Thelipophilic moiety is attached to the amine group of the N-terminalserine. The amine group of the serine forms an amide bond with thecarboxyl of a 3,4-dihydroxytetradecanoyl moiety in pseudomycin A, a3-monohydroxytetradecanoyl moiety in pseudomycin B, a3,4-dihydroxyhexadecanoyl moiety in pseudomycin C and a3-monohydroxyhexadecanoyl moiety in pseudomycin C′. The carboxyl groupof the serine forms an amide bond with the Dab of the ring.

[0011] Pseudomycins A′ and B′

[0012] As used herein the terms pseudomycin A′ and pseudomycin B′ referto antifungal agents that have been isolated from the bacteriumPseudomonas syringae. Pseudomycins A′ and B′ are pseudomycins having thecharacteristic depsinonapeptide ring with the sequenceSer-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr, with the carboxyl group of theClThr and the hydroxyl group of the serine closing the ring with alactone bond. Pseudomycin A′ includes a 3,4-dihydroxypentadecanoic acidmoiety, the carboxyl group of which forms an amide bond with the aminegroup of the N-terminal serine. Pseudomycin B′ includes a3-hydroxydodecanoic acid moiety, the carboxyl group of which forms anamide bond with the amine group of the N-terminal serine.

[0013] Biological Activities of Pseudomycins

[0014] A pseudomycin has several biological activities including killingvarious fungi, such as fungal pathogens of plants and animals. Inparticular, a pseudomycin is an active antimycotic agent against fungithat cause opportunistic infections in immune compromised individuals.These fungi include various species of Candida including C.parapsilosis, C. albicans, C. glabrata, C. tropicalis, and C. krusei.They also incldue other genera such as Cryptococcus neoformans,Aspergillus fumigatus, and Histoplasma capsulatum. Killing, rather thaninhibiting the growth of fungi, particularly of fungal pathogens, is adesirable and preferred biological activity of an antifungal, such aspseudomycin A′ and/or B′.

[0015] The pseudomycins have been shown to be toxic to a broad range ofplant-pathogenic fungi including Rynchosporium secalis, Ceratocystisulmi, Rizoctonic solani, Sclerotinia sclerotiorum, Verticilliumalbo-atrum, Verticillium dahliae, Thielaviopis basicola, Fusariumoxysporum and Fusarium culmorum. (see Harrison. L. et al.,“Pseudomycins, a family of novel peptides from Pseudomonas syringaepossessing broad-spectrum antifungal activity,” J. of GeneralMicrobiology, 7, 2857-2865 (1991).) In addition, P. syringae MSU 16H hasbeen shown to confer a greater protection than the wild-type strain inelms infected with Ceratocystic ulmi, the causal agent of Dutch elmdisease, (see e.g., Lam et al. Proc. Natl. Sci. USA, 84, 6447-6451(1987)).

[0016]Pseudomonas syringae

[0017]Pseudomonas syringae include a wide range of bacteria that aregenerally associated with plants. Some of the P. syringae are plantpathogens, while others are only weakly pathogenic or are saprophytes.Many different isolates of P. syringae produce one or more cytotoxicagents that can help this bacterium survive in the wild where it mustcompete with fungi and other bacteria. The cytotoxic agents produced byP. syringae include anti-fungal agents such as the pseudomycins, thesyringomycins, the syringotoxins, and the syringostatins.

[0018] Strains of P. syringae that produce one or more pseudomycins havebeen described in the art. For example, wild type strain MSU 174(isolated from a Montana barley field) and a mutant of this straingenerated by transposon mutagenesis using TN905 (MSU 16H) are describedin U.S. Pat. No. 5,576,298, issued Nov. 19, 1996 to G. Strobel et al.:Harison et al., J. “Pseudomycins, a family of novel peptides fromPseudomonas syringae possessing broad-spectrum antifungal activity,”Gen. Microbiology 137, 2857-2865 (1991): and Lamb et al., “Transposonmutagenesis and tagging of fluorescent pseudomonas: Antimycoticproduction is necessary for control of Dutch elm disease,” Proc. Natl.Acad. Sci. USA 84, 6447-6451 (1987). Methods for growth of variousstrains of P. syringae and their use in production of antifungal agentssuch as pseudomycins are also disclosed in U.S. patent application Ser.No. ________ by Matthew D. Hilton et al, entitled “PseudomycinProduction By Pseudomonas Syringae” submitted evendate herewith anddescribed below. Cultures of MSU 174 and MSU 16H are on deposit atMontana State University (Bozeman, Mo. USA) and available from theAmerican Type Culture Collection (Parklawn Drive, Rockville, Md. USA).The disclosures of the references cited in this paragraph areincorporated herein by reference.

[0019] The present invention includes a strain, an isolate, and abiologically-purified culture of P. syringae that produces pseudomycinA′ and/or B′, in amounts at least about 10 μg/mL. Preferably, thebiologically-purified culture of a microorganism is of Pseudomonassyringae strain MSU 16H, 25-B1, 67H1, or 7H9-1, or a mutant, variant,isolate, or recombinant of these strains that produces pseudomycin Aand/or B′. Cultures MSU 174 and MSU 16H were obtained as described inthe references cited herein above.

[0020] A strain of P. syringae that is suitable for production ofpseudomycin A′ and/or B′ can be isolated from environmental sourcesincluding plants, such as barley plants, citrus plants, and lilacplants, and also from sources such as soil, water, air, and dust. Apreferred strain is isolated from plants. Strains of P. syringae thatare isolated from environmental sources can be referred to as wild type.As used herein, “wild type” refers to a dominant genotype whichnaturally occurs in the normal population of P. syringae (i.e., strainsor isolates of P. syringae that are found in nature and not produced bylaboratory manipulation). As is the case with other organisms, thecharacteristics of the pseudomycin A′ and/or B′ producing culturesemployed in this invention, P. syringae strains such as MSU 174, MSU16H, MSU 206, 25-B1, 7H9-1, and 67 H1 are subject to variation. Thus,progeny of these strains, e.g., recombinants, mutants and variants, maybe obtained by methods well-known to those skilled in the art.

[0021] Mutant strains of P. syringae are also suitable for production ofpseudomycin A′ and/or B′. As used herein, mutant refers to a suddenheritable change in the phenotype of a strain, which can be spontaneousor induced by known mutagenic agents, including radiation and variouschemicals. Mutant P. syringae of the present invention can be producedusing a variety of mutagenic agents including radiation such asultraviolet light, x-rays; chemical mutagens, site-specific mutagenesis,and transposon mediated mutagenesis. Examples of chemical mutagens areethyl methanesulfonate (EMS), diepoxyoctane,N-methyl-N-nitro-N′-nitrosoguanine (NTG), and nitrous acid.

[0022] Pseudomycin A′ and/or B′ producing mutants of P. syringae of thepresent invention can be produced by treating the bacteria with anamount of a mutagenic agent effective to produce mutants thatoverproduce pseudomycin A′ and/or B′, that produce pseudomycin A′ and/orB′ in excess over other pseudomycins, or that produce pseudomycin A′and/or B′ under advantageous growth conditions. While the type andamount of mutagenic agent to be used can vary, a preferred method is toserially dilute NTG to levels ranging from 1 to 100 μg/ml. Preferredmutants of the invention are those that overproduce pseudomycin A′and/or B′ grow in minimal defined media. The mutants overproducepseudomycin A′ and/or B′ preferably to at least about 10 μg/mL.

[0023] Environmental isolates, mutant strains, and other desirablestrains of P. syringae can be subjected to selection for desirabletraits of growth habit, growth medium, nutrient source, carbon source,growth conditions, and amino acid requirements. Preferably, apseudomycin A′ and/or B′ producing strain of P. syringae is selected forgrowth on minimal defined medium, such as N21 medium, and/or forproduction pseudomycin A and/or B′ at levels greater than about 10μg/mL. Preferred strains exhibit the characteristic of producingpseudomycin A′ and/or B′ when grown on a medium including glycine and,optionally, either a lipid, a potato product, or a combination thereof.

[0024] Recombinant strains can be developed by transforming the P.syringae strains, using established laboratory procedures well-known tothose skilled in the art. Through the use of recombinant technology, theP. syringae strains can be transformed to express a variety of geneproducts in addition to the antibiotics these strains produce. Forinstance, one can transform the strains with a recombinant vector thatconfers resistance to an antibiotic to which the strains are normallysensitive. Transformants thus obtained will produce not onlypseudomycins, such as pseudomycins A′ and/or B′, but also theresistance-conferring enzyme that allows selection of the transformedfrom wild-type cells. Furthermore, using similar techniques, one canmodify the present strains to introduce multiple copies of theendogenous pseudomycin-biosynthesis genes to achieve greaterpseudomycin, such as pseudomycin A′ and/or B′ yield, Progeny, i.e.natural and induced variants, mutants and recombinants, of the P.syringae strains 25-B 1, 67H1, and 7H9-1 which retain the characteristicof pseudomycin, such as pseudomycin A′ and/or B′ overproduction are partof this invention.

[0025] Growth of Pseudomonas syringae

[0026] As described herein, “aqueous nutrient media” refers to awater-base composition including minerals and organic compounds andtheir salts necessary for growth of the bacterium used in the presentinvention. Preferred nutrient media contain an effective amount of threeor fewer amino acids, preferably, glutamic acid, glycine, histidine or acombination thereof. In one embodiment, the medium contains an effectiveamount of glycine and, optionally, one or more of a potato product and alipid. Glycine can be provided as a single amino acid or as part of amixture of amino acids, such as hydrolyzed protein. Suitable lipidsinclude soybean oil, or a fatty acid. Suitable potato products includepotato dextrose broth, potato dextrin, potato protein, and commercialmashed potato mix food product. Preferred minerals in the nutrientmedium include salt mixtures typically used in cell culture andfermentation, such as Czapek mineral salts solution (e.g., KCl, MgSO₄,and FeSO₄). The organic compound in the nutrient media preferablyincludes glucose and can optionally include soluble starch: other likeorganic compounds can also be included. The pH of the medium ispreferably between about 4 and 6.5, more preferably about 4.5 to about5.7, most preferably about 5.2.

[0027] Although the amount of each ingredient in the nutrient broth isnot typically critical to growth of the bacteria or to production ofpseudomycin A′ and/or B′ certain levels of nutrients are advantageous. Apreferred amount of glycine is about 0.1 g/L to about 10 g/L, morepreferably about 0.3 g/L to about 3 g/L, most preferably about 1 g/L. Apreferred amount of lipid is about 1 g/L to about 10 g/L of an oilproduct such as soybean oil, more preferably about 0.5 g/L to about 2g/L of soybean oil. A preferred amount of a fatty acid or fatty acidester is about 0.5 g/L to about 5 g/L. Preferred amounts of potatoproducts include about 12 g/L to about 36 g/L preferably about 24 g/L ofpotato dextrose broth; about 5 g/L to about 50 g/L, preferably about 30g/L of commercial mashed potato mix; about 1 g/L to about 30 g/L,preferably about 20 g/L of potato dextrin, or about 1 g/L to about 10g/L, preferably about 4 g/L of potato protein. A preferred nutrientmedium includes minerals, preferably, KCl at about 0.02 to about 2 g/L,more preferably about 0.2 g/L: MgSO₄, preferably MgSO₄.7H₂O, at about0.02 to about 2 g/L, more preferably about 0.2 g/L; and FeSO₄,preferably FeSO₄.7H₂O, at about 0.4 to about 40 mg/L, more preferablyabout 4 mg/L. When present, soluble starch is preferably at about 0.5 toabout 50 g/L, more preferably about 5 g/L. Glucose is preferably presentat about 2 to about 80 g/L, more preferably about 20 g/L.

[0028]P. syringae are typically grown in the media described underconditions of controlled or regulated pH, and temperature, P. syringaegrow and pseudomycin A′ and/or B′ at temperatures between about 15° C.and about 35° C., preferably about 20° C. to about 30° C. morepreferably about 22° C. to about 27° C., most preferably about 25° C. P.syringae grow and produce pseudomycin A′ and/or B′ at pH between about 4and about 9, preferably about 4 and about 6, more preferably about 4.5to about 5.5. Typically growth of P. syringae does not occur when thetemperature is above 37° C. or below 10° C. or when the pH is above 9 orbelow 4.

[0029] Method for Production of Pseudomycins A′ and B′

[0030] To produce pseudomycin A′ and/or B′ from a wild type or mutantstrain of P. syringae, the organism is cultured with agitation in anaqueous nutrient medium including an effective amount of three or feweramino acids. The three or fewer amino acids are preferably glutamicacid, glycine, histidine, or a combination thereof. In one preferredembodiment, the amino acids include glycine and, optionally, one or moreof a potato product and a lipid. Culturing is conducted under conditionseffective for growth of P. syringae and production of pseudomycin A′and/or B′. Effective conditions include temperature of about 22° C. toabout 27° C., and a duration of about 36 hours to about 96 hours. Whencultivated on the media such as those described herein, P. syringae cangrow in cell densities up to about 10-15 g/L dry weight and producepseudomycins A′ and/or B′ in total amounts at least about 10 μg/mL.

[0031] Controlling the concentration of oxygen in the medium duringculturing of P. syringae is advantageous for production of pseudomycinA′ and/or B′. Preferably, oxygen levels are maintained at about 5% toabout 50% saturation, preferably about 30% saturation. Sparging withair, with pure oxygen, or with gas mixtures including oxygen canregulate the concentration of oxygen in the medium. Further, adjustmentof the agitation rate can be used to adjust the oxygen transfer rate.

[0032] Controlling the pH of the medium during culturing of P. syringaeis advantageous for production of a pseudomycin A′ and/or B′.Pseudomycins, such as pseudomycins A′ and/or B′, are labile at basic pH,and significant degradation can occur if the pH of the culture medium isabove about 6 for more than about 12 hours. Preferably, the pH of theculture medium is maintained at less than about 6, preferably less thanabout 5.5, and preferably above 4.0. The pH is preferably maintained atabout 5 to about 5.4, more preferably about 5.0 to about 5.2. Althoughnot limiting to the present invention, it is believed that pseudomycindegradation at basic pH is due to opening of the lactone ring andconversion of ClThr to Thr.

[0033]P. syringae can produce pseudomycins A and/or B′ when grown inbatch culture. However, fed-batch or semi-continuous feed of glucoseand, optionally, an acid or base, such as ammonium hydroxide, to controlpH, enhances pseudomycin production. Pseudomycin production by P.syringae can be further enhanced by using continuous culture methods inwhich glucose and, optionally, an acid or base, such as ammoniumhydroxide, to control pH, are fed automatically.

[0034] Pseudomycins A′ and/or B′ can be detected, determined, isolated,and/or purified by any of a variety of methods known to those of skillin the art. For example, the level of pseudomycin activity in a broth orin an isolated or purified composition can be determined by antifungalaction against a fungus such as Candida. Numerous methods are known forthe preparation and analysis of the pseudomycins. For example, one ormore pseudomycins can be isolated and purified by chromatography, suchas HPLC.

Pharmaceutical Uses

[0035] Formulations and Antifungal Action of Pseudomycin A′ or B′0

[0036] Each of pseudomycin A′ and B′ show in vitro and in vivo activityand therefore may useful in combating either systemic fungal infectionsor fungal skin infections. Accordingly, the present invention provides amethod of inhibiting fungal activity including contacting pseudomycin A′and/or B′ or a pharmaceutically acceptable salt thereof with a fungus. Apreferred method includes inhibiting growth or activity of various fungiincluding C. parapsilosis, C. albicans, Cryptococcus neoformans, andHistoplasma capsulatum. As used herein contacting a compound of theinvention with a parasite or fungus refers to a union or junction, orapparent touching or mutual tangency of a compound of the invention witha parasite or fungus. However, the term contacting does not imply anymechanism of inhibition.

[0037] The present invention further provides a method of treating afungal infection which includes administering an effective amount ofpseudomycin A′ and/or B′, or a pharmaceutically acceptable salt,hydrate, or ester thereof, to a host in need of such treatment. Apreferred method includes treating an infection by various fungiincluding C. parapsilosis. C. albicans, Cryptococcus neoformans, andHistoplasma capsulatum. When administered in an effective andappropirate amount, a formulation of pseudomycin A′ and/or B′ reducesthe burden of a fungal infection, reduces symptoms associated with thefungal infection, and can result in the elimination of the fungalinfection.

[0038] Some patients in need of antifungal therapy have severe symptomsof infection, such as high fever, and are likely to be in intensive orcritical care. Various fungi can cause such serious infections. Candidaspp., for example, may cause mucosal and serious systemic infections.Azole and polyene resistant strains of Candida have been reported withincreasing frequency. Aspergillus causes life-threatening systemicinfections. Cryptococcus is responsible for meningitis. Such seriousfungal infections may occur in immune compromised patients, such asthose receiving organ or bone marrow transplants, undergoingchemotherapy for cancer, recovering from major surgery, or sufferingfrom HIV infection. For such patients, antifungal therapy wouldtypically include intravenous administration of a formulation containingpseudomycin A′ and/or B′ over several days or more to halt theinfection.

[0039] With respect to antifungal activity, the term “effective amount,”means an amount of a compound of the present invention which is capableof inhibiting fungal growth or activity, or reducing symptoms of thefungal infection. For most fungal infections reduction of symptoms ofthe infection includes reduction of fever, return to consciousness, andincreased well being of the patient. Preferably, symptoms are reduced bykilling the fungus to eliminate the infection or to bring the infectionto a level tolerated by the patient or controlled by the patient'simmune system. As used herein inhibiting refers to inhibiting fungalactivity, including stopping, retarding or prophylactically hindering orpreventing the growth or any attending characteristics and results fromthe existence of a fungus.

[0040] The dose administered will vary depending on such factors as thenature and severity of the infection, the age and general health of thehost and the tolerance of the host to the antifungal agent. Typically,the compositions will be administered to a patient (human or otheranimal, including mammals such as, but not limited to, cats, horses andcattle and avian species) in need thereof, in an effective amount toinhibit the fungal infection. The particular dose regimen likewise mayvary according to such factors and may be given in a single daily doseor in-multiple doses during the day. The regimen may last from about 2-3days to about 2-3 weeks or longer. A typical daily dose (administered insingle or divided doses) will contain a dosage level of from about 0.01mg/kg to about 100 mg/kg of body weight of an active compound of thisinvention. Preferred daily doses generally will be from about 0.1 mg/kgto about 60 mg/kg and ideally from about 2.5 mg/kg to about 40 mg/kg.For serious infections, the compound can be administered by intravenousinfusion using, for example, 0.01 to 10 mg/kg/hr of the activeingredient.

[0041] The present invention also provides pharmaceutical formulationsuseful for administering the antifungal compounds of the invention.Accordingly, the present invention also provides a pharmaceuticalformulation including one or more pharmaceutically acceptable carriers,diluents, vehicles, excipients, or other additives and pseudomycin A′and/or B′. The active ingredient in such formulations includes from 0.1%to 99.9% by weight of the formulation, more generally from about 10% toabout 30% by weight. By “pharmaceutically acceptable” it is meant thatthe carrier, diluent or excipient is compatible with the otheringredients of the formulation and not deleterious to the recipientthereof.

[0042] The formulation can include additives such as various oils,including those of petroleum, animal, vegetable or synthetic origin, forexample, peanut oil, soybean oil, mineral oil, and sesame oil. Suitablepharmaceutical excipients include starch, cellulose, glucose, lactose,sucrose, gelatin, malt, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk, glycerol, propyleneglycol, water, and ethanol. The compositions can be subjected toconventional pharmaceutical expedients, such as sterilization, and cancontain conventional pharmaceutical additives, such as preservatives,stabilizing agents, wetting, or emulsifying agents, salts for adjustingosmotic pressure, and buffers. Suitable pharmaceutical carriers andtheir formulations are described in Martin. “Remington's PharmaceuticalSciences,” 15 th Ed.: Mack Publishing Co., Easton (1975): see. e.g., pp.1405-1412 and pp. 1461-1487.

[0043] The term “pharmaceutically acceptable salt”, as used herein,refers to salts of the compounds described above that are substantiallynon-toxic to living organisms. Typical pharmaceutically acceptable saltsinclude those salts prepared by reaction of the compounds of the presentinvention with a mineral or organic acid or an inorganic base. Suchsalts are known as acid addition and base addition salts.

[0044] Acids commonly employed to form acid addition salts are mineralacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, and phosphoric acid, and organic acids such asp-toluenesulfonic, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, and acetic acid. Examples of such pharmaceuticallyacceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite,bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,propionate, decanoate, caprylate, acrylate, formate, isobutyrate,caproate, heptanoate, propiolate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,napththalene-2-sulfonate, and mandelate. Preferred pharmaceuticallyacceptable acid addition salts are those formed with mineral acids suchas hydrochloric acid and hydrobromic acid, and those formed with organicacids such as maleic acid and methanesulfonic acid.

[0045] Base addition salts include those derived from inorganic bases,such as ammonium or alkali or alkaline earth metal hydroxides,carbonates, and bicarbonates. Such bases useful in preparing the saltsof this invention thus include sodium hydroxide, potassium hydroxide,ammonium hydroxide, potassium carbonate, sodium carbonate, sodiumbicarbonate, potassium bicarbonate, calcium hydroxide, and calciumcarbonate. The potassium and sodium salt forms are particularlypreferred.

[0046] It should be recognized that the particular counterion forming apart of any salt of this invention is not of a critical nature, so longas the salt as a whole is pharmacologically acceptable and as long asthe counterion does not contribute undesired qualities to the salt as awhole.

[0047] Pseudomycin A′ and/or B′ may be administered parenterally, forexample using intramuscular, subcutaneous, or intraperitoneal injection,nasal, or oral routes. In addition to these methods of administration,pseudomycin A′ and/or B′ may be applied topically for superficial skininfections, or eradication or inhibition of fungi in the mucus.

[0048] For parenteral administration the formulation includespseudomycin A′ and/or B′ and a physiologically acceptable diluent suchas deionized water, physiological saline, 5% dextrose and other commonlyused diluents. The formulation may contain a cyclodextrin and/or asolubilizing agent such as a polyethylene glycol or polypropylene glycolor other known solubilizing agent. Such formulations may be made up insterile vials containing the antifungal and excipient in a dry powder orlyophilized powder form. Prior to use, a physiologically acceptablediluent is added and the solution withdrawn via syringe foradministration to the patient.

[0049] The present pharmaceutical formulations arm prepared by knownprocedures using known and readily available ingredients. In making thecompositions of the present invention, the active ingredient willgenerally be admixed with a carrier, or diluted by a carrier, orenclosed within a carrier which may be in the form of a capsule, sachet,paper or other container. When the carrier serves as a diluent, it maybe a solid, semi-solid or liquid material which acts as a vehicle,excipient or medium for the active ingredient. Thus, the compositionscan be in the form of tablets, pills, powders, lozenges, sachets,cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols,(as a solid or in a liquid medium), ointments containing, for example,up to 10% by weight of the active compound, soft and hard gelatincapsules, suppositories, sterile injectable solutions, and sterilepackaged powders.

[0050] For oral administration, the antifungal compound is filled intogelatin capsules or formed into tablets. Such tablets may also contain abinding agent, a dispersant or other suitable excipients suitable forpreparing a proper size tablet for the dosage pseudomycin A′ and/or B′.For pediatric or geriatric use the antifungal compound may be formulatedinto a flavored liquid suspension, solution or emulsion. A preferredoral formulation is linoleic acid, cremophor RH-60 and water andpreferably in the amount (by volume) of 8% linoleic acid, 5% cremophorRH-60. 87% sterile water and pseudomycin A′ and/or B′ in an amount offrom about 2.5 to about 40 mg/ml.

[0051] For topical use the antifungal compound may be formulated with adry powder for application to the skin surface or it may be formulatedin a liquid formulation including a solubilizing aqueous liquid ornon-aqueous liquid, e.g., an alcohol or glycol.

[0052] Uses of Formulations of Pseudomycin A′ or B′

[0053] The present invention also encompasses a kit including thepresent pharmaceutical compositions and to be used with the methods ofthe present invention. The kit can contain a vial which contains aformulation of the present invention and suitable carriers, either driedor in liquid form. The kit further includes instructions in the form ofa label on the vial and/or in the form of an insert included in a box inwhich the vial is packaged, for the use and administration of thecompounds. The instructions can also be printed on the box in which thevial is packaged. The instructions contain information such assufficient dosage and administration information so as to allow a workerin the field to administer the drug. It is anticipated that a worker inthe field encompasses any doctor, nurse, or technician who mightadminister the drug.

[0054] The present invention also relates to a pharmaceuticalcomposition including a formulation of pseudomycin A′ and/or B′ and thatis suitable for administration by injection. According to the invention,a formulation of pseudomycin A′ and/or B′, can be used for manufacturinga composition or medicament suitable for administration by injection.The invention also relates to methods for manufacturing compositionsincluding a formulation of pseudomycin A′ and/or B′ in a form that issuitable for administration by injection. For example, a liquid or solidformulation can be manufactured in several ways, using conventionaltechniques. A liquid formulation can be manufactured by dissolvingpseudomycin A′ and/or B′, in a suitable solvent, such as water, at anappropriate pH, including buffers or other excipients.

Agricultural Uses

[0055] Antibiotics produced from P. syringae NRRL B-12050 have beendemonstrated to effectively treat Dutch elm disease. (see, e.g., U.S.Pat. Nos. 4,342,746 and 4,277,462) In particular. P. syringae MSU 16Hhas been shown to confer a greater protection than the wild-type strainin elms infected with Ceratocystis ulmi, the causal agent of Dutch elmdisease. (see e.g., Lam et al. Proc. Natl. Sci. USA. 84. 6447-46451(1987)). More extensive tests on field-grown elms confirmed thephenomenon of biocontrol at the prophylactic level. Hence, thepseudomycins of the present invention may be useful as a preventativetreatment for Dutch Elm disease. The pseudomycins have been shown to betoxic to a broad range of plant-pathogenic fungi including Rynchosporiumsecalis, Ceratocystis ulmi, Rizoctonia solani, Sclerotinia sclerotiorum,Verticillium albo-atrum, Verticillium dahliae, Thielaviopis basicola,Fusarium oxysporum and Fusarium culmorum. (see Harrison. L., et al.,“Pseudomycins, a family of novel peptides from Pseudomonas syringaepossessing broad-spectrum antifungal activity.” J. General Microbiology,7, 2857-2865 (1991).) Consequently, the isolated pseudomycin A′ and/orB′ (including hydrates, solvates, and esters thereof) may be useful inthe treatment of fungi in plants (in particular, V. albo-atrum,Rhizoctonia solani and F. oxysprum) either as a direct treatment orpreventative treatment. Generally, the infected plants are treated byinjecting or spraying an aqueous suspension of the pseudomycin compoundsinto or onto the plant. Means of injection are well-known to thoseskilled in the art (e.g., gouge pistol). Any means of spraying thesuspension may be used that distributes an effective amount of theactive material onto the plant surface. The suspension may include otheradditives generally used by those skilled in the art, such assolubilizers, stabilizers, wetting agents, and combinations thereof.

[0056] Treatment of the plant may also be accomplished using a drycomposition containing the isolated pseudomycin A′ and/or B′ compounds.The dry formulation may be applied to the plant surface by any meanswell-known to those skilled in the art, such as spraying or shaking froma container.

[0057] The present invention may be better understood with reference tothe following examples. These examples are intended to be representativeof specific embodiments of the invention, and are not intended aslimiting the scope of the invention.

EXAMPLES Biological Materials on Deposit

[0058]P. syringae MSU 16H is publicly available from the American TypeCulture Collection, Parklawn Drive, Rockville, Md. USA as Accession No.ATCC 67028. P. syringae strains 25-B1. 7H9-1. and 67 H1 were depositedwith the American Type Culture Collection on Mar. 23, 2000 and wereassigned the following Accession Nos.: 25-B1 Accession No. PTA-16227H9-1 Accession No. PTA-1623 67 H1 Accession No. PTA-1621

Example 1 Production of Pseudomycins A′ and B′

[0059] Fermentation methods were developed for producing pseudomycin A′and/or B′ in the fermentation broth of a Pseudomonas syringae strain.

[0060] Materials and Methods

[0061] Preparation of inoculum: An aliquot of cells stored in the vaporphase of liquid nitrogen was thawed and used to inoculate two 900 mLportions of CSM broth. CSM broth was composed of (g/L): dextrose (5),maltose (4), Difco Tryptic Soy Broth (30). Difco yeast extract (3), andMgSO₄ 7H₂O (2). Approximately 0.5 mL of cells was used to inoculate each900 mL portion of medium contained in a two liter flask. Flasks wereincubated with shaking for 24 hours at 25° C. The contents of two flaskswere combined to inoculate a 150 liter fermentor containing 115 litersof sterile fermentation broth.

[0062] Fermentation Stage: Fermentation broth was composed of (g/L):dextrose (20), soluble starch (5), Basic American Foods Country StylePotato Pearls instant mashed potatoes (30), glycine (1), MgSO₄ 7H₂O(0.2), KCl (0.2), and FeSO₄ 7H₂O (0.004) in tap water. The pH wasadjusted to 5.2 before sterilization. Fermentation was carried out at25° C. for 68 hr. Dissolved oxygen was maintained at or above 30% of airsaturation by continuous adjustment of air flow and impeller agitationrate. The pH was maintained between 4.0 and 5.4 through the addition ofeither H₂SO₄ or NaOH.

[0063] Variations on the Batch Methods: Several variations of the simplebatch process were also found to produce pseudomycins A′ and/or B′.Dextrose can be fed to the fermentors starting 24 hours after initialinoculation at a rate of 60 mL per hour. Feeding can be continuedthroughout the course of the fermentation. Alternatively, a process hasbeen used where dissolved oxygen is maintained at 5% of air saturationstarting 24 hours after inoculation and continuing until the end of thefermentation period. Maintenance of dissolved oxygen at 5% was achievedthrough addition of inert nitrogen gas (N₂) to the air supply leading tothe fermentor. In all cases, gas was supplied through a single submergedsparger tube with an opening positioned just below the bottom agitatorturbine in the fermentor.

[0064] Results and Conclusions

[0065] Several fermentation methods produce pseudomycin A′ and/or B′from P. syringae.

Example 2 Isolation and Purification of Pseudomycins A′ and B′

[0066] Methods were developed for isolation and purificationpseudomycins A′ and B′ from the fermentation broth of a Pseudomonassyringae strain.

[0067] Materials and Methods

[0068] The whole fermentation broth (4×100 L) after harvest was filteredthrough a Membralox ceramic filter (0.45 μm) to afford a filtrate(fraction A) and a solid slurry (fraction B). Fraction B (135 L) wasextracted with an equal volume of acetone containing 0.1% TFA for 120min and allowed to settle. The clear acetone extract was separated byfiltration and then evaporated in vacuo to an aqueous solution to yieldfraction C (88 L). First, fraction A was charged on to a HP20ss resincolumn (10 L) packed in water and the column was washed with 15%acetonitrile containing 0.1% TFA (20 L). Fraction C was then loaded onto the same column and the column was washed with 20 L of 15%acetonitrile containing 0.1% TFA as before.

[0069] The column was then eluted with a linear gradient of 15-20%acetonitrile containing 0.1% TFA over 30 min and 20-35% acetonitrilecontaining 0.1% TFA over 60 min with 1 L/min flow rate. One literfractions were collected. Fractions 6-9 were combined (4 L) to yieldfraction D (24 g). A portion of fraction D (˜1 g) was chromatographedover a reversed-phase column (Dynamax C₁₈ 41.4×250 mm) usingtriethylammonium phosphate buffer (pH 3)-acetonitrile-methanol as mobilephase (65:17:18 to 30:35:35 gradient elution over 45 min with 40 ml/minflow rate). Appropriate fractions were combined, volume was reduced to75 ml and rechromatographed over a C₁₈ column as before using a gradient80:10:10 to 46:27:27 to afford fraction E (113 mg) and fraction F (116mg). Further chromatography of fractions E and F over a C₁₈ column(Dynamax 21.4×250 mm) furnished 45 mg pseudomycin A′ and 62 mg ofpseudomycin B′, respectively.

[0070] Results and Conclusions

[0071] HPLC methods similar to those used to purify other pseudomycinsresulted in purification of pseudomycins A′ and B′ from fermentationbroth.

Example 3 Determination of the Structure of Pseudomycins A′ and B′

[0072] Mass spectrometry and NMR determined the structures pseudomycinsA′ and B′.

[0073] Structure Determination of Pseudomycin A′

[0074] Methods and Results

[0075] The molecular formula of pseudomycin A′ was determined by highresolution FABMS as C₅₂H₈₉ClN₁₂O₂₀ [m/z 1237.6112 for C₅₂H₉₀ClN₁₂O₂₀(M+H)⁺, Δ-2.4 ppm]. When compared to pseudomycin A the molecular formulaof pseudomycin A′ showed one additional CH₂ group. This observationsuggested that in pseudomycin A′ the N-terminal serine may be acylatedwith 3,4-dihydroxypentadecanoic acid instead of3,4-dihydroxytetradecanoic acid as in pseudomycin A. This argument issupported by the fact that in all previously characterized pseudomycins,the core possess a distinctive and identical nonadepsipeptide ring andthe only difference among them arise due to the nature of thehydrophobic side chain.

[0076] Accordingly the NMR spectral data of pseudomycin A′ is virtuallyidentical to all the known pseudomycins, such as pseudomycin A, B, C andC′. Comprehensive analysis of ¹H, ¹³C. and 2D NMR spectra includingTOCSY and HMQC of pseudomycin A′ established 3,4 diol functionality inthe hydrophobic side chain of pseudomycin A′ and enabled assignment ofall the protons and proton bearing carbons in the molecule (Table 1).The structure determined for pseudomycin A′ based on these massspectrometric and NMR data is shown below.

[0077] Structure of Pseudomycin A′ Derived from Mass and NMR SpectralData TABLE 1 ¹H and ¹³CNMR data of Pseudomycin A' in H₂O + CD₃CN Aminoacid Position δ_(H) δ_(C) Ser NH 8.28 — α 4.59 54.0 β1 4.50 65.5 β2 4.41Dab-1* NH 8.48 — α 4.15 53.1 β1 1.98 γ 2.91 37.4 NH₂ 7.50 — Asp NH 8.34— α 4.54 51.5 β1 2.86 36.0 β2 2.80 Lys NH 7.80 — α 4.16 54.6 β 1.75 30.8γ1 1.31 23.2 γ2 1.22 δ 1.54 27.2 ε 2.83 40.4 NH₂ 7.34 — Dab-2* NH 8.09 —α 4.28 52.1 β1 2.11 28.7 β2 1.96 γ 2.89 37.6 NH₂ — Thr NH 7.63 — α 4.2859.8 β 3.92 68.6 γ 1.16 20.4 Dhb NH 9.45 — β 6.49 133.9 γ 1.69 13.5 Hyd.Asp NH 7.85 — α 4.94 56.9 β 4.78 71.6 C1Thr NH 7.88 α 4.87 56.0 β 4.3172.3 γ1 3.50 45.6 γ2 3.42 Side chain 2a 2.47 39.4 2b 2.30 3 3.76 72.6 43.39 75.1 5 1.41 33.3 6-14 1.21 32.4, 30.2X4, 29.9, 27.2, 26.4, 23.2 150.81 14.3

[0078] Structure Determinati n f Pseudomycin B′

[0079] The structure determination of pseudomycin B′ was againaccomplished through the interpretation of mass and NMR spectral data.The molecular formula C₄₉H₈₃ClN₁₂O₁₉ [m/z 1179.5685 for C₄₉H₈₄ClN₁₂O₁₉(M+H)⁺, Δ-1.8 ppm] was established by high resolution FAB-MS data. Thisformula showed two CH₂ less than that observed for pseudomycin B.Detailed analysis of ¹H, ¹³C and 2D NMR including TOCSY and HMQC spectraand comparison of the spectral data with those of known pseudomycinsagain revealed identical amino acid composition. In addition the NMRdata indicated the presence of 3-hydroxydodecanoic acid (Table 2). Thus,from this spectral data, the structure of pseudomycin B′ is derived asshown below.

[0080] Structure of Pseudomycin B′ Derived from Mass and NMR SpectralData TABLE 2 ¹H and ¹³CNMR data of Pseudomycin B' in H₂O + CD₃CN Aminoacid Position δ_(H) δ_(C) Ser NH 8.31 — α 4.64 53.5 β1 4.54 65.8 β2 4.35Dab-1* NH 8.52 — α 4.13 53.3 β1 2.02 28.7 β2 γ 2.94 37.3 NH₂ 7.54 — AspNH 8.30 — α 4.56 51.6 β1 2.86 36.0 β2 2.80 Lys NH 7.90 — α 4.09 54.9 β1.75 29.8 γ1 1.28 23.2 γ2 1.18 δ 1.52 27.3 ε 2.82 40.4 NH₂ 7.34 — Dab-2*NH 8.24 α 4.35 51.8 β1 2.12 29.2 β2 1.99 γ 2.90 37.7 NH₂ — Thr NH 7.75 —α 4.23 60.4 β 3.93 68.2 γ 1.18 20.5 Dhb NH 9.45 — β 6.57 134.8 γ 1.6813.7 Hyd. Asp NH 7.79 — α 4.95 57.1 β 4.71 72.0 C1Thr NH 7.98 α 4.8755.8 β 4.31 72.5 γ1 3.48 45.6 γ2 3.42 Side chain 2a 2.33 43.8 2b 2.24 33.85 69.6 4 1.37 37.6 5-11 1.20 32.4, 30.1, 30.1, 29.8, 23.2 12 0.8114.4

[0081] Conclusions

[0082] Pseudomycins A′ and B′ represent new members of a unique class ofnonadepsipeptides. Although these molecules are very closely related tothe known pseudomycins differing only in the nature of the hydrophobicside chain, they should play a key role in elucidating thestructure-activity relationship among this class of compounds asantifungals.

Example 4 Isolation, Characterization and Mutagenesis of Pseudomonassyringae

[0083] Environmental isolates and mutants of P. syringae were producedand employed in production of antifungal agents.

[0084] Materials and Methods

[0085] Strains MSU 174 and MSU 16-H were isolated and characterized asdescribed in U.S. Pat. No. 5,576,298, issued Nov. 19, 1996 to G. Strobelet al.: Harrison et al., “Pseudomycins, a family of novel peptides fromPseudomonas syringae possessing broad-spectrum antifungal activity.” J.Gen. Microbiology 137, 2857-2865 (1991); and Lamb et al., “Transposonmutagenesis and tagging of fluorescent pseudomonas: Antimycoticproduction is necessary for control of Dutch elm disease.” Proc. Natl.Acad. Sci. USA 84, 6447-6451 (1987). The disclosures of the referencescited in this paragraph are incorporated herein by reference.

[0086] Additional strains were derived from such wild type andtransposon generated mutants by chemical mutagenesis. Strains subjectedto mutagenesis include MSU 174. MSU 16H, and 25-B 1. The strain to bemutagenized was grown in CSM medium, then divided into the mediumincluding 0, 1, 2, 4, 16, or 32 μg/mL of the chemical mutagen1-methyl-3-nitro-1-nitrosoguanidine (NTG or MNNG). These cells were thenfrozen for future screening and selection.

[0087] Mutagenized cells were selected for desirable growth conditionsand/or production of one or more Pseudomycins, such as pseudomycin A′and/or B′. Chemically mutagenized cells of P. syringae, such asmutagenized strain 25-B 1, were thawed and diluted to 6 cells/mL inN21SM medium (Table 5). This medium sometimes contained one or morecomponents for selection, such as varying concentrations of phosphate. A50 μL volume of mutagenized cells was dispensed into a well of a 96-wellround bottom microtiter plate for a delivery of an average of 0.3cells/well. Typically, silicone oil was added to each well to minimizeevaporation. The plates were incubated with shaking for 6 to 12 days at25° C. TABLE 5 The Composition of N21SM Medium GRAMS INGREDIENT PERLITER Glucose 20 Ammonium Sulfate 0.5 Monosodium Glutamate or L-glutamicacid 2 L-Histidine 2 Glycine 0.5 Soluble Starch 5 KH₂PO₄ 0.2 CzapekMineral Salts Solution 2 mL MES Buffer 9.8 Adjust pH to 5.0

[0088] After this incubation, an aliquot, typically 5 TL, from each wellwas serially diluted (e.g. 1:56.1:196, 1:320, 1:686, and/or 1:1715) andevaluated for activity against Candida albicans in a liquid microtiterplate bioassay. The plates were incubated at 37° C. overnight and thewells were scored for inhibition of C. albicans growth. Suitable strainswere picked, inoculated into CSM medium (Table 6), and grown for 1 to 3days at 25° C. TABLE 6 Complete Streptomyces Medium (CSM) ComponentConcentration (g/L) Glucose 5 Maltose 4 Difco Tryptic Soy Broth 30 DifcoYeast Extract 3 MgSO₄.7H₂O 2 No pH adjustment

[0089] The selected strains were preserved and inoculated intofermentation bottles containing 13 mL of N21SM medium and grown forapproximately 66 hours at 25° C. An aliquots was removed from thisfermentation, extracted for 1 hour with a volume of acetonitrile equalto the volume of the aliquot, centrifuged, and decanted for HPLCanalysis of one or more Pseudomycins, such as pseudomycin A′ or B′, asdescribed in Examples 1-3. Strains producing one or more Pseudomycins,such as pseudomycin A′ or B′, were reisolated, refermented, and preparedfor growth on a larger scale.

[0090] Results

[0091] Strains exhibiting production of one or more Pseudomycins, suchas pseudomycin A′ or B′, were produced using the methods describedabove.

[0092] Conclusion

[0093] The selection methods and criteria disclosed herein are effectivefor producing strains of P. syringae that grow on minimal medium andproduce one or more pseudomycins, such as pseudomycin A′ or B′.

Example 5 Gr wth of P. syringae and Production f Pseud mycins

[0094] Fermentation of P. syringae in medium N21, which does not includeany potato products used in published media for growth of P. syringae,produced pseudomycins at levels suitable for isolation.

[0095] Production of Pseudomycins in Shaken Flasks and N21 Medium

[0096] Materials and Methods

[0097]P. syringae were grown in 50 mL of N21 culture medium (Table 7) ina 250 mL flask. The culture was started with an aliquot of an inoculumof P. syringae MSU 16H and maintained at 25° C. and 70% humidity for 7days with shaking at 250 rpm in an incubator. At the end of theincubation period 4 mL of the broth are removed from the flask and mixedwith 6 mL of methanol containing 0.1% phosphoric acid. It is believedthat low pH stabilizes the pseudomycins. Particulate matter is removedby filtration or centrifugation and the pseudomycins were determined byHPLC as described herein. TABLE 7 The Composition of N21 Medium GRAMSINGREDIENT PER LITER Sucrose 35 Ammonium Sulfate 0.5 MonosodiumGlutamate 2 L-Histidine 2 Glycine 0.5 Soluble Starch 5 KH₂PO₄ 0.2 CzapekMineral Salts Solution 2 mL Yeast Extract 1 MES Buffer 9.8 Adjust pH to5.2

[0098] Production of pseudomycin by several strains of P. syringae wasevaluated employing N21 medium with and without added methyl myristate.The strains of P. syringae evaluated included MSU 16H, 25-B1, 67H1, and7H9- 1.

[0099] Results

[0100] The various strains of P. syringae when grown with or withoutmethyl myristate produced significant levels of one or morepseudomycins, for example more than 10 Tg/mL of one or more ofpseudomycin A, pseudomycin B, and/or pseudomycin C. Methyl myristatestimulated pseudomycin production for certain strains.

[0101] Conclusions

[0102] Various strains of P. syringae produce commercially significantlevels of pseudomycins in medium lacking potato products, and thisproduction can be stimulated by methyl myristate.

[0103] Production f Pseudomycins at a Scale of 5,000 L Employing N21Medium

[0104] The methods for producing pseudomycins employing a medium withoutadded potato products was scaled up to a 5,000 liter level.

[0105] Materials and Methods

[0106] Vegetative-stage flasks containing complete streptomyces medium(CSM, Table 5) inoculated with a frozen P. syringae culture, typicallystrain 67H1, and were shaken at 250 rpm and 25° C. for 24 hours. After24 hours of incubation of the vegetative-stage flasks, the contents ofthese flasks was used to inoculate bump-stage flasks. The bump-stageflasks included the CSM medium and were rotated at 250 rpm and held at25° C. The bump-stage flasks were inoculated with about 0.45 mL ofpooled culture from three or four vegetative-stage flasks. The bumpstage flasks typically include about 900 mL of CSM in a non-baffled 2.5L Tunair™ flask. Two bump-stage cultures in Tunair™ flasks were set foreach fermentor. The bump-stage flasks were incubated for 16 hours.

[0107] Then, two of the bump-stage cultures were pooled by combining inan inoculation bazooka. These combined cultures were used to inoculate atank containing the medium described in Table 15 that has beensupplemented with an additional 3 g/L (for a total of 4 g/L) of glycine,1 g/L of soybean oil, and 1 g/L of yeast extract. These large-scalecultures were grown at 25° C. for three to four days. During thisgrowing period, dissolved oxygen was controlled at 30% of air saturationwith agitation and air flow, pH was controlled at 5.2±0.2 by addition ofsulfuric acid or sodium hydroxide as required. Eighteen hours afterbeginning the large-scale culture, a glucose feed was started at a rateof 200 mL/h. Twenty hours after the start of the large-scale culture,ammonium hydroxide feed was started at a rate of 20 mL/h. During thisculture, the holdback pressure was 5 psig. The initial setting foragitation was 150 rpm and air flow is 0.5 scfm. If required, ananti-foam agent was added, as well. Certain variations on theseconditions were tested as well. After the three to four days oflarge-scale culture, the P. syringae were harvested. Antifungal activitywas measured as described in Example 6.

[0108] Results and Conclusions

[0109] Pseudomycins were produced in commercially significant amounts atthe 5,000 L scale employing a medium free of added potato products.

Example 6 Determination and Purification of Pseudomycins

[0110] Detection and Quantification of Pseudomycins by AntifungalActivity

[0111] The presence or amount of a pseudomycin or mixture ofpseudomycins can be determined by measuring the antifungal activity of apreparation. Antifungal activity was determined in vitro by obtainingthe minimum inhibitory concentration (MIC) of the preparation using astandard agar dilution test or a disc-diffusion test. A preparation ofone or more pseudomycins can be an extract of a cell culture, or a morepurified mixture. A typical fungus employed in testing antifungalactivity is C. albicans. Antifungal activity was considered significantwhen the test preparation caused 10-12 mm diameter zones of inhibitionon Candida albicans x657 seeded agar plates.

[0112] The antifungal studies were conducted using a microtiter brothdilution assay according to National Committee for Clinical LaboratoryStandards guidelines in 96 well microtiter plates. Sabourauds anddextrose broth was adjusted to contain 2.5×10⁴ conida/ml. Test compoundwas dissolved in water and tested in two-fold dilutions starting withthe highest concentration of 20 μg/ml. Plates were incubated at 35° C.for 48 hr. The results in Table 3 and 4 show the minimal inhibitoryconcentration (MIC) of the compound that completely inhibited growthcompared to untreated growth controls. TABLE 3 Antifungal Activity ofPseudomycin A′ Organism MIC (μ/ml) Candida albicans 2.5 C. parapsilosis5.0 Cryptococcus neoformans 1.25 Aspergillus fumigatus >20 Histoplasmacapsulatum 5.0

[0113] TABLE 4 Antifungal Activity of Pseudomycin B′ Organism MIC (μ/ml)Candida albicans 10 C. parapsilosis 10 Cryptococcus neoformans 1.25Aspergillus fumigatus >20 Histoplasma capsulatum 1.25-5.0

[0114] Detection and Quantification of Pseudomycins by HPLC

[0115] A sample believed to contain one or more pseudomycins wasclarified by filtration or centrifugation. The clarified mixture waschromatographed on a Zorbax RxC8 column (3.5 T particles 25×0.46 cm)with a flow rate of 1 ml/min. The column was eluted with 20-55%acetonitrile with 0.2% TFA linear gradient over 15 min and held at 55%acetonitrile with 0.2% TFA for 5 min. Typically, pseudomycin A′ elutedat about 13.7 min. (822 sec) and pseudomycin B′ eluted at about 12.4 min(822 sec). Pseudomycins were detected by absorbance at 215 nm andquantified by integration of UV peaks. A standard of each of thepseudomycins was employed for identification and quantification.

Example 7 Formulations Including Pseudomycin A′ and/or B′

[0116] The following formulation examples are illustrative only and arenot intended to limit the scope of the invention in any way. The term“active ingredient” means pseudomycin A′ and/or B′ or a pharmaceuticallyacceptable salt thereof.

[0117] Formulation 1

[0118] Hard gelatin capsules are prepared using the followingingredients: Quantity Ingredient (mg/capsule) Active ingredient 250Starch, dried 200 Magnesium stearate 10 Total 460 mg

[0119] Formulation 2

[0120] A tablet is prepared using the ingredients below. The componentsare blended and compressed to form tablets each weighing 665 mg.Quantity Ingredient (mg/capsule) Active ingredient 250 Cellulose,microcrystalline 400 Silicon dioxide, fumed 10 Stearic acid 5 Total 665mg

[0121] Formulation 3

[0122] An aerosol solution is prepared containing the followingcomponents. The active compound is mixed with ethanol and the mixtureadded to a portion of the propellant 22. cooled to −30° C. andtransferred to a filling device. The required amount is then fed to astainless steel container and diluted with the remainder of thepropellant. The valve units are then fitted to the container. ComponentWeight (g) Active ingredient 0.25 Methanol 27.75 Propellant 22 74.00(Chlorodifluoromethane) Total 100.00

[0123] Formulation 4

[0124] Tablets, each containing 60 mg of active ingredient, are made asfollows: Active ingredient  60 mg Microcrystalline cellulose  45 mgPolyvinylpyrrolidone (as 10%  4 mg solution in water) Sodiumcarboxymethyl starch  4.5 mg Magnesium stearate  0.5 mg Talc  1 mg Total150 mg

[0125] The active ingredient, starch and cellulose are passed through aNo. 45 mesh U.S. sieve and mixed thoroughly. The aqueous solutioncontaining polyvinyl-pyrrolidone is mixed with the resultant powder, andthe mixture then is passed through a No. 14 mesh U.S. sieve. Thegranules so produced are dried at 50° C. and passed through a No. 18mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate andtalc, previously passed through a No. 60 mesh U.S. sieve, are then addedto the granules which, after mixing, are compressed on a tablet machineto yield tablets each weighing 150 mg.

[0126] Formulation 5

[0127] Capsules, each containing 80 mg of active ingredient, are made asfollows: Active ingredient  80 mg Starch  59 mg Microcrystallinecellulose  59 mg Magnesium stearate  2 mg Total 200 mg

[0128] The active ingredient, cellulose, starch and magnesium stearateare blended, passed through a No. 45 mesh U.S. sieve, and filled intohard gelatin capsules in 200 mg quantities.

[0129] Formulation 6

[0130] Suppositories, each containing 225 mg of active ingredient, aremade as follows: Active ingredient   225 mg Saturated fatty acidglycerides 2.000 mg Total 2.225 mg

[0131] The active ingredient is passed through a No. 60 mesh U.S. sieveand suspended in the saturated fatty acid glycerides previously meltedusing the minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

[0132] Formulation 7

[0133] Suspensions, each containing 50 mg of active ingredient per 5 mldose, are made as follows: Active ingredient   50 mg Sodiumcarboxymethyl   50 mg cellulose Syrup 1.25 ml Benzoic acid solution 0.10ml Flavor q.v. Color q.v. Purified water to total   5 ml

[0134] The active ingredient is passed through a No. 45 mesh U.S. sieveand mixed with the sodium carboxymethyl cellulose and syrup to form asmooth paste. The benzoic acid solution, flavor and color are dilutedwith a portion of the water and added, with stirring. Sufficient wateris then added to produce the required volume.

[0135] Formulation 8

[0136] An intravenous formulation may be prepared as follows. Thesolution of these ingredients generally is administered intravenously toa subject at a rate of 1 ml per minute. Active ingredient   100 mgIsotonic saline 1,000 mg

[0137] The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

We claim:
 1. An isolated pseudomycin natural product comprising apseudomycin, a pseudomycin B′, a mixture thereof, or a pharmaceuticallyacceptable salt, hydrate or ester thereof.
 2. The pseudomycin of claim1, comprising pseudomycin A′ or a pharmaceutically acceptable salt,hydrate or ester thereof.
 3. The pseudomycin of claim 2, having theformula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 4. Thepseudomycin of claim 1, comprising pseudomycin B′ or a pharmaceuticallyacceptable salt, hydrate or ester thereof.
 5. The pseudomycin of claim4, having the formula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 6. Anisolated compound having the formula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 7. Anisolated compound having the formula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 8. Amethod of inhibiting fungal activity comprising contacting a fungus withan isolated pseudomycin natural product comprising a pseudomycin A′, apseudomycin B′, a mixture thereof, or a pharmaceutically acceptablesalt, hydrate or ester thereof.
 9. The method of claim 8, wherein thepseudomycin natural product comprises pseudomycin A′ or apharmaceutically acceptable salt, hydrate or ester thereof.
 10. Themethod of claim 9, wherein the pseudomycin natural product has theformula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 11. Themethod of claim 8, wherein the pseudomycin natural product comprisespseudomycin B′, or a pharmaceutically acceptable salt, hydrate or esterthereof.
 12. The method of claim 11, wherein the pseudomycin naturalproduct has the formula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 13. Themethod of claim 8, wherein the fungus comprises Candida parapsilosis,Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum.14. A method of inhibiting fungal activity comprising contacting afungus with an isolated compound having the formula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 15. Amethod of inhibiting fungal activity comprising contacting a fungus withan isolated compound having the formula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 16. Amethod of reducing the symptoms of a fungal infection in a patient inneed thereof comprising: administering to the patient an effectiveamount of a composition comprising an isolated pseudomycin naturalproduct comprising a pseudomycin A′, a pseudomycin B′, a mixturethereof, or a pharmaceutically acceptable salt, hydrate or esterthereof.
 17. The method of claim 16, wherein the pseudomycin naturalproduct comprises pseudomycin A′, or a pharmaceutically acceptable salt,hydrate or ester thereof.
 18. The method of claim 17, wherein thepseudomycin natural product has the formula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 19. Themethod of claim 16, wherein the pseudomycin natural product comprisespseudomycin B′ or a pharmaceutically acceptable salt, hydrate or esterthereof.
 20. The method of claim 19, wherein the pseudomycin naturalproduct has the formula:

or a pharmaceutically acceptable salt, hydrate or ester thereof.
 21. Themethod of claim 16, wherein the fungus comprises Candida parapsilosis,Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum.22. The use of the compounds of claims 1, 2, 3, 4, 5, 6 or 7 in themanufacture of a pharmaceutical composition for inhibiting fungalactivity.
 23. The use of the compounds of claims 1, 2, 3, 4, 5, 6 or 7in the manufacture of a pharmaceutical composition for inhibiting fungalactivity or reducing the symptoms of a fungal infection in a patient.